The interest for producing wheat flour with health promoting effect and improved functionality has led to investigate new milling techniques that can provide finer flours. In this study, jet milling treatment was used to understand the effect of ultrafine size reduction onto microstructure and physicochemical properties of wheat flour. Three different conditions of jet milling, regarding air pressure (4 or 8 bars) feed rate and recirculation, were applied to obtain wheat flours with different particle size (control, F1, F2 and F3 with d50 127.45, 62.30, 22.94 and 11.4 μm, respectively). Large aggregates were gradually reduced in size, depending on the intensity of the process, and starch granules were separated from the protein matrix. Damaged starch increased while moisture content decreased because of milling intensity. Notable changes were observed in starch hydrolysis kinetics, which shifted to higher values with milling. Viscosity of all micronized samples was reduced and gelatinization temperatures (To, Tp, Tc) for F2 and F3 flours increased. Controlling jet milling conditions allow obtaining flours with different functionality, with greater changes at higher treatment severity that induces large particle reduction.